Nucleoside phosphoramidates

ABSTRACT

A nucleoside compound having activity against hepatitis C virus is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/645,765, filed Dec. 23, 2009, which claims priority to U.S.Provisional Patent Application No. 61/140,423, filed Dec. 23, 2008, thecontents of which are incorporated by reference in entirety herein.

INCORPORATION OF SEQUENCE LISTING

The Sequence Listing filed on Feb. 25, 2013, created on May 10, 2012,named 03956054400_ST25.txt, having a size in bytes of 953 B, is herebyincorporated by reference in its entirety.

FIELD OF INVENTION

The present invention pertains to nucleoside phosphoramidates and theiruse as agents for treating viral diseases. These compounds areinhibitors of RNA-dependent RNA viral replication and are useful asinhibitors of HCV NS5B polymerase, as inhibitors of HCV replication andfor treatment of hepatitis C infection in mammals.

BACKGROUND

Hepatitis C virus (HCV) infection is a major health problem that leadsto chronic liver disease, such as cirrhosis and hepatocellularcarcinoma, in a substantial number of infected individuals, estimated tobe 2-15% of the world's population. There are an estimated 4.5 millioninfected people in the United States alone, according to the U.S. Centerfor Disease Control. According to the World Health Organization, thereare more than 200 million infected individuals worldwide, with at least3 to 4 million people being infected each year. Once infected, about 20%of people clear the virus, but the rest can harbor HCV the rest of theirlives. Ten to twenty percent of chronically infected individualseventually develop liver-destroying cirrhosis or cancer. The viraldisease is transmitted parenterally by contaminated blood and bloodproducts, contaminated needles, or sexually and vertically from infectedmothers or carrier mothers to their offspring. Current treatments forHCV infection, which are restricted to immunotherapy with recombinantinterferon-α alone or in combination with the nucleoside analogribavirin, are of limited clinical benefit. Moreover, there is noestablished vaccine for HCV. Consequently, there is an urgent need forimproved therapeutic agents that effectively combat chronic HCVinfection.

The HCV virion is an enveloped positive-strand RNA virus with a singleoligoribonucleotide genomic sequence of about 9600 bases which encodes apolyprotein of about 3,010 amino acids. The protein products of the HCVgene consist of the structural proteins C, E1, and E2, and thenon-structural proteins NS2, NS3, NS4A and NS4B, and NS5A and NS5B. Thenonstructural (NS) proteins are believed to provide the catalyticmachinery for viral replication. The NS3 protease releases NS5B, theRNA-dependent RNA polymerase from the polyprotein chain. HCV NS5Bpolymerase is required for the synthesis of a double-stranded RNA from asingle-stranded viral RNA that serves as a template in the replicationcycle of HCV. Therefore, NS5B polymerase is considered to be anessential component in the HCV replication complex (K. Ishi, et al,Heptology, 1999, 29: 1227-1235; V. Lohmann, et al., Virology, 1998, 249:108-118). Inhibition of HCV NS5B polymerase prevents formation of thedouble-stranded HCV RNA and therefore constitutes an attractive approachto the development of HCV-specific antiviral therapies.

HCV belongs to a much larger family of viruses that share many commonfeatures.

Flaviviridae Viruses

The Flaviviridae family of viruses comprises at least three distinctgenera: pestiviruses, which cause disease in cattle and pigs;flaviviruses, which are the primary cause of diseases such as denguefever and yellow fever; and hepaciviruses, whose sole member is HCV. Theflavivirus genus includes more than 68 members separated into groups onthe basis of serological relatedness (Calisher et al., J. Gen. Virol,1993,70,37-43). Clinical symptoms vary and include fever, encephalitisand hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe,D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia,Pa., 1996, Chapter 31, 931-959). Flaviviruses of global concern that areassociated with human disease include the Dengue Hemorrhagic Feverviruses (DHF), yellow fever virus, shock syndrome and Japaneseencephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6,251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., NewEng. J. Med, 1988, 319, 64 1-643).

The pestivirus genus includes bovine viral diarrhea virus (BVDV),classical swine fever virus (CSFV, also called hog cholera virus) andborder disease virus (BDV) of sheep (Moennig, V. et al. Adv. Vir. Res.1992, 41, 53-98). Pestivirus infections of domesticated livestock(cattle, pigs and sheep) cause significant economic losses worldwide.BVDV causes mucosal disease in cattle and is of significant economicimportance to the livestock industry (Meyers, G. and Thiel, H. J.,Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al., Adv.Vir. Res. 1992, 41, 53-98). Human pestiviruses have not been asextensively characterized as the animal pestiviruses. However,serological surveys indicate considerable pestivirus exposure in humans.

Pestiviruses and hepaciviruses are closely related virus groups withinthe Flaviviridae family. Other closely related viruses in this familyinclude the GB virus A, GB virus A-like agents, GB virus-B and GBvirus-C (also called hepatitis G virus, HGV). The hepacivirus group(hepatitis C virus; HCV) consists of a number of closely related butgenotypically distinguishable viruses that infect humans. There are atleast 6 HCV genotypes and more than 50 subtypes. Due to the similaritiesbetween pestiviruses and hepaciviruses, combined with the poor abilityof hepaciviruses to grow efficiently in cell culture, bovine viraldiarrhea virus (BVDV) is often used as a surrogate to study the HCVvirus.

The genetic organization of pestiviruses and hepaciviruses is verysimilar. These positive stranded RNA viruses possess a single large openreading frame (ORF) encoding all the viral proteins necessary for virusreplication. These proteins are expressed as a polyprotein that is co-and post-translationally processed by both cellular and virus-encodedproteinases to yield the mature viral proteins. The viral proteinsresponsible for the replication of the viral genome RNA are locatedwithin approximately the carboxy-terminal. Two-thirds of the ORF aretermed nonstructural (NS) proteins. The genetic organization andpolyprotein processing of the nonstructural protein portion of the ORFfor pestiviruses and hepaciviruses is very similar. For both thepestiviruses and hepaciviruses, the mature nonstructural (NS) proteins,in sequential order from the amino-terminus of the nonstructural proteincoding region to the carboxy-terminus of the ORF, consist of p7, NS2,NS3, NS4A, NS4B, NS5A, and NS5B.

The NS proteins of pestiviruses and hepaciviruses share sequence domainsthat are characteristic of specific protein functions. For example, theNS3 proteins of viruses in both groups possess amino acid sequencemotifs characteristic of serine proteinases and of helicases (Gorbalenyaet al., Nature, 1988, 333, 22; Bazan and Fletterick Virology, 1989,171,637-639; Gorbalenya et al., Nucleic Acid Res., 1989, 17, 3889-3897).Similarly, the NS5B proteins of pestiviruses and hepaciviruses have themotifs characteristic of RNA-directed RNA polymerases (Koonin, E. V. andDolja, V. V., Crir. Rev. Biochem. Molec. Biol. 1993, 28, 375-430).

The actual roles and functions of the NS proteins of pestiviruses andhepaciviruses in the lifecycle of the viruses are directly analogous. Inboth cases, the NS3 serine proteinase is responsible for all proteolyticprocessing of polyprotein precursors downstream of its position in theORF (Wiskerchen and Collett, Virology, 1991, 184, 341-350;Bartenschlager et al., J. Virol. 1993, 67, 3835-3844; Eckart et al.Biochem. Biophys. Res. Comm. 1993, 192,399-406; Grakoui et al., J.Virol. 1993, 67, 2832-2843; Grakoui et al., Proc. Natl. Acad. Sci. USA1993, 90, 10583-10587; Hijikata et al., J. Virol. 1993, 67, 4665-4675;Tome et al., J. Virol., 1993, 67, 4017-4026). The NS4A protein, in bothcases, acts as a cofactor with the NS3 serine protease (Bartenschlageret al., J. Virol. 1994, 68, 5045-5055; Fulla et al., J. Virol. 1994, 68,3753-3760; Xu et al., J. Virol., 1997, 71:53 12-5322). The NS3 proteinof both viruses also functions as a helicase (Kim et al., Biochem.Biophys. Res. Comm., 1995, 215, 160-166; Jin and Peterson, Arch.Biochem. Biophys., 1995, 323, 47-53; Warrener and Collett, J. Virol.1995, 69,1720-1726). Finally, the NS5B proteins of pestiviruses andhepaciviruses have the predicted RNA-directed RNA polymerases activity(Behrens et al., EMBO, 1996, 15, 12-22; Lechmann et al., J. Virol.,1997, 71, 8416-8428; Yuan et al., Biochem. Biophys. Res. Comm. 1997,232, 231-235; Hagedorn, PCT WO 97/12033; Thong et al, J. Virol., 1998,72, 9365-9369).

Currently, there are limited treatment options for individuals infectedwith hepatitis C virus. The current approved therapeutic option is theuse of immunotherapy with recombinant interferon-α alone or incombination with the nucleoside analog ribavirin. This therapy islimited in its clinical effectiveness and only 50% of treated patientsrespond to therapy. Therefore, there is significant need for moreeffective and novel therapies to address the unmet medical need posed byHCV infection.

A number of potential molecular targets for drug development of directacting antivirals as anti-HCV therapeutics have now been identifiedincluding, but not limited to, the NS2-NS3 autoprotease, the N3protease, the N3 helicase and the NS5B polymerase. The RNA-dependent RNApolymerase is absolutely essential for replication of thesingle-stranded, positive sense, RNA genome and this enzyme has elicitedsignificant interest among medicinal chemists.

Inhibitors of HCV NS5B as potential therapies for HCV infection havebeen reviewed: Tan, S.-L., et al., Nature Rev. Drug Discov., 2002, 1,867-881; Walker, M. P. et al., Exp. Opin. Investigational Drugs, 2003,12, 1269-1280; Ni, Z-J., et al., Current Opinion in Drug Discovery andDevelopment, 2004, 7, 446-459; Beaulieu, P. L., et al., Current Opinionin Investigational Drugs, 2004, 5, 838-850; Wu, J., et al., Current DrugTargets-Infectious Disorders, 2003, 3, 207-219; Griffith, R. C., et al,Annual Reports in Medicinal Chemistry, 2004, 39, 223-237; Carrol, S., etal., Infectious Disorders-Drug Targets, 2006, 6, 17-29. The potentialfor the emergence of resistant HCV strains and the need to identifyagents with broad genotype coverage supports the need for continuingefforts to identify novel and more effective nucleosides as HCV NS5Binhibitors.

Nucleoside inhibitors of NS5B polymerase can act either as a non-naturalsubstrate that results in chain termination or as a competitiveinhibitor which competes with nucleotide binding to the polymerase. Tofunction as a chain terminator the nucleoside analog must be taken up bythe cell and converted in vivo to a triphosphate to compete for thepolymerase nucleotide binding site. This conversion to the triphosphateis commonly mediated by cellular kinases which imparts additionalstructural requirements on a potential nucleoside polymerase inhibitor.Unfortunately, this limits the direct evaluation of nucleosides asinhibitors of HCV replication to cell-based assays capable of in situphosphorylation.

In some cases, the biological activity of a nucleoside is hampered byits poor substrate characteristics for one or more of the kinases neededto convert it to the active triphosphate form. Formation of themonophosphate by a nucleoside kinase is generally viewed as the ratelimiting step of the three phosphorylation events. To circumvent theneed for the initial phosphorylation step in the metabolism of anucleoside to the active triphosphate analog, the preparation of stablephosphate prodrugs has been reported. Nucleoside phosphoramidateprodrugs have been shown to be precursors of the active nucleosidetriphosphate and to inhibit viral replication when administered to viralinfected whole cells (McGuigan, C., et al., J. Med. Chem., 1996, 39,1748-1753; Valette, G., et al., J. Med. Chem., 1996, 39, 1981-1990;Balzarini, J., et al., Proc. National Acad Sci USA, 1996, 93, 7295-7299;Siddiqui, A. Q., et al., J. Med. Chem., 1999, 42, 4122-4128; Eisenberg,E. J., et al., Nucleosides, Nucleotides and Nucleic Acids, 2001, 20,1091-1098; Lee, W. A., et al., Antimicrobial Agents and Chemotherapy,2005, 49, 1898); US 2006/0241064; and WO 2007/095269.

Also limiting the utility of nucleosides as viable therapeutic agents istheir sometimes poor physicochemical and pharmacokinetic properties.These poor properties can limit the intestinal absorption of an agentand limit uptake into the target tissue or cell. To improve on theirproperties prodrugs of nucleosides have been employed. It has beendemonstrated that preparation of nucleoside phosphoramidates improvesthe systemic absorption of a nucleoside and furthermore, thephosphoramidate moiety of these “pronucleotides” is masked with neutrallipophilic groups to obtain a suitable partition coefficient to optimizeuptake and transport into the cell dramatically enhancing theintracellular concentration of the nucleoside monophosphate analogrelative to administering the parent nucleoside alone. Enzyme-mediatedhydrolysis of the phosphate ester moiety produces a nucleosidemonophosphate wherein the rate limiting initial phosphorylation isunnecessary.

SUMMARY OF THE INVENTION

The present invention is directed to a phosphoramidate nucleosiderepresented by formula I, or its stereoisomers, salts, pharmaceuticallyacceptable salts, hydrates, solvates, crystalline, or metabolite formsthereof:

Z is

wherein when

-   -   Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is OMe, N(—CH₂CH₂CH₂—) OBn, or OH; and R⁹ is NH₂;

and wherein when

-   -   Z is

R5 is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe, —N(—CH₂CH₂CH₂—)(azetidin-1-yl), OBn, or OH; and R⁹ is NH₂.

DEFINITIONS

The phrase “a” or “an” entity as used herein refers to one or more ofthat entity; for example, a compound refers to one or more compounds orat least one compound. As such, the terms “a” (or “an”), “one or more”,and “at least one” can be used interchangeably herein.

The phrase “as defined herein above” or “as defined herein” refers tothe first definition provided in the Summary of the Invention.

The terms “optional” or “optionally” as used herein means that asubsequently described event or circumstance may but need not occur, andthat the description includes instances where the event or circumstanceoccurs and instances in which it does not. For example, “optional bond”means that the bond may or may not be present, and that the descriptionincludes single, double, or triple bonds.

The term “independently” is used herein to indicate that a variable isapplied in any one instance without regard to the presence or absence ofa variable having that same or a different definition within the samecompound. Thus, in a compound in which R appears twice and is defined as“independently carbon or nitrogen”, both R's can be carbon, both R's canbe nitrogen, or one R′ can be carbon and the other nitrogen.

The term “purified,” as described herein, refers to the purity of agiven compound. For example, a compound is “purified” when the givencompound is a major component of the composition, i.e., at least 50% w/wpure. Thus, “purified” embraces at least 50% w/w purity, at least 60%w/w purity, at least 70% purity, at least 80% purity, at least 85%purity, at least 90% purity, at least 92% purity, at least 94% purity,at least 96% purity, at least 97% purity, at least 98% purity, and atleast 99% purity.

It is also contemplated that the compound represented by formula Iembraces deuterated analogs. The term “deuterated analogs” means acompound described herein or its salts thereof, whereby a ¹H-isotope,i.e., hydrogen (H), is substituted by a ²H-isotope, i.e., deuterium (D).Deuterium substitution can be partial or complete. Partial deuteriumsubstitution means that at least one hydrogen is substituted by at leastone deuterium. For instance, for a compound represented by formula II,one of ordinary skill can contemplate at least the following partialdeuterated analogs (where “d_(n)” represents n-number of deuteriumatoms, such as, for an isopropyl group n=1-7, while for a phenyl group,n=1-5). Although the methyl groups depicted below are shown as beingcompletely deuterated, one will recognize that partial-deuteratedvariations are also possible, such as, —CDH₂ and —CD₂H.

These are but a few deuterated analogs that are synthetically accessibleby procedures and reagents that are known to one of ordinary skill.

The term “metabolite,” as described herein, refers to a compoundproduced in vivo after administration to a subject in need thereof.

The term “salts,” as described herein, refers to a compound produced bythe protonation of a proton-accepting moiety and/or deprotonation of aproton-donating moiety. It should be noted that protonation of theproton-accepting moiety results in the formation of a cationic speciesin which the charge is balanced by the presence of a physiologicalanion, whereas deprotonation of the proton-donating moiety results inthe formation of an anionic species in which the charge is balanced bythe presence of a physiological cation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a phosphoramidate nucleosiderepresented by formula I, or its stereoisomers, salts, pharmaceuticallyacceptable salts, hydrates, solvates, crystalline, or metabolite formsthereof:

Z is

wherein when

-   -   Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is OMe, —N(—CH₂CH₂CH₂—) (azetidin-1-yl), OBn, orOH; and R⁹ is NH₂;

and wherein when

-   -   Z is

R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe, —N(—CH₂CH₂CH₂—)(azetidin-1-yl), OBn, or OH; and R⁹ is NH₂.

A first embodiment of the present invention is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

wherein when

-   -   Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is OMe; and R⁹ is NH₂;

and wherein when

-   -   Z is

R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe; and R⁹ is NH₂.

A first aspect of the first embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is OMe; and R⁹ is NH₂.

A second aspect of the first embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ismethyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; andR⁸ is OMe; and R⁹ is NH₂.

A third aspect of the first embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ismethyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe; and R⁹ is NH₂.

A fourth aspect of the first embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ is^(i)Pr; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe; and R⁹ is NH₂.

A fifth aspect of the first embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ iscyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe; and R⁹ isNH₂.

A sixth aspect of the first embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ishydrogen; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe; and R⁹ isNH₂.

A seventh aspect of the first embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

R¹ is hydrogen; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ishydrogen; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe; and R⁹ isNH₂.

An eighth aspect of the first embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

wherein

-   -   Z is

and wherein R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OMe; and R⁹ isNH₂.

A second embodiment of the present invention is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

wherein when

-   -   Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is —N(—CH₂CH₂CH₂—) (azetidin-1-yl); and R⁹ isNH₂;

and wherein when

-   -   Z is

R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is —N(—CH₂CH₂CH₂—)(azetidin-1-yl); and R⁹ is NH₂.

A first aspect of the second embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is —N(—CH₂CH₂CH₂—) (azetidin-1-yl); and R⁹ isNH₂.

A second aspect of the second embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ismethyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; andR⁸ is —N(—CH₂CH₂CH₂—) (azetidin-1-yl); and R⁹ is NH₂.

A third aspect of the second embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ismethyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is —N(—CH₂CH₂CH₂—)(azetidin-1-yl); and R⁹ is NH₂.

A fourth aspect of the second embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ is^(i)Pr; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is —N(—CH₂CH₂CH₂—)(azetidin-1-yl); and R⁹ is NH₂.

A fifth aspect of the second embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ iscyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is—N(—CH₂CH₂CH₂—) (azetidin-1-yl); and R⁹ is NH₂.

A sixth aspect of the second embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ishydrogen; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is —N(—CH₂CH₂CH₂—)(azetidin-1-yl); and R⁹ is NH₂.

A seventh aspect of the second embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

R¹ is hydrogen; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ishydrogen; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is —N(—CH₂CH₂CH₂—)(azetidin-1-yl); and R⁹ is NH₂.

An eighth aspect of the second embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

wherein

-   -   Z is

and wherein R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is —N(—CH₂CH₂CH₂—)(azetidin-1-yl); and R⁹ is NH₂.

A third embodiment of the present invention is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

wherein when

-   -   Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is OBn; and R⁹ is NH₂;

and wherein when

-   -   Z is

R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OBn; and R⁹ is NH₂.

A first aspect of the third embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is OBn; and R⁹ is NH₂.

A second aspect of the third embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ismethyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; andR⁸ is OBn; and R⁹ is NH₂.

A third aspect of the third embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ismethyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OBn; and R⁹ is NH₂.

A fourth aspect of the third embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ is^(i)Pr; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OBn; and R⁹ is NH₂.

A fifth aspect of the third embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ iscyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OBn; and R⁹ isNH₂.

A sixth aspect of the third embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ishydrogen; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OBn; and R⁹ isNH₂.

A seventh aspect of the third embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

R¹ is hydrogen; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ishydrogen; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OBn; and R⁹ isNH₂.

An eighth aspect of the third embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

wherein

-   -   Z is

and wherein R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OBn; and R⁹ isNH₂.

A fourth embodiment of the present invention is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

wherein when

-   -   Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is OH; and R⁹ is NH₂;

and wherein when

-   -   Z is

R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OH; and R⁹ is NH₂.

A first aspect of the fourth embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) isCH₃; R⁴ is hydrogen, methyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶is CH₃; X is F; and R⁸ is OH; and R⁹ is NH₂.

A second aspect of the fourth embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ismethyl, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; andR⁸ is OH; and R⁹ is NH₂.

A third aspect of the fourth embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ismethyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OH; and R⁹ is NH₂.

A fourth aspect of the fourth embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ is^(i)Pr; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OH; and R⁹ is NH₂.

A fifth aspect of the fourth embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ iscyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OH; and R⁹ isNH₂.

A sixth aspect of the fourth embodiment is directed to a phosphoramidatenucleoside represented by formula I, or its stereoisomers, salts,pharmaceutically acceptable salts, hydrates, solvates, crystalline, ormetabolite forms thereof:

Z is

R¹ is phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ishydrogen; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OH; and R⁹ isNH₂.

A seventh aspect of the fourth embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

Z is

R¹ is hydrogen; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ ishydrogen; R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OH; and R⁹ isNH₂.

An eighth aspect of the fourth embodiment is directed to aphosphoramidate nucleoside represented by formula I, or itsstereoisomers, salts, pharmaceutically acceptable salts, hydrates,solvates, crystalline, or metabolite forms thereof:

wherein

-   -   Z is

and wherein R⁵ is hydrogen; R⁶ is CH₃; X is F; and R⁸ is OH; and R⁹ isNH₂.

Dosage, Administration, and Use

A fifth embodiment of the present invention is directed to a compositionfor the treatment of any of the viral agents disclosed herein saidcomposition comprising a pharmaceutically acceptable medium selectedfrom among an excipient, carrier, diluent, or equivalent medium and acompound, that is intended to include its salts (acid or basic additionsalts), hydrates, solvates, and crystalline forms can be obtained,represented by formula I.

It is contemplated that the formulation of the fifth embodiment cancontain any of the compounds contemplated in the present inventioneither alone or in combination with another compound of the presentinvention.

The compounds of the present invention may be formulated in a widevariety of oral administration dosage forms and carriers. Oraladministration can be in the form of tablets, coated tablets, hard andsoft gelatin capsules, solutions, emulsions, syrups, or suspensions.Compounds of the present invention are efficacious when administered bysuppository administration, among other routes of administration. Themost convenient manner of administration is generally oral using aconvenient daily dosing regimen which can be adjusted according to theseverity of the disease and the patient's response to the antiviralmedication.

A compound or compounds of the present invention, as well as theirpharmaceutically acceptable salts, together with one or moreconventional excipients, carriers, or diluents, may be placed into theform of pharmaceutical compositions and unit dosages. The pharmaceuticalcompositions and unit dosage forms may be comprised of conventionalingredients in conventional proportions, with or without additionalactive compounds and the unit dosage forms may contain any suitableeffective amount of the active ingredient commensurate with the intendeddaily dosage range to be employed. The pharmaceutical compositions maybe employed as solids, such as tablets or filled capsules, semisolids,powders, sustained release formulations, or liquids such as suspensions,emulsions, or filled capsules for oral use; or in the form ofsuppositories for rectal or vaginal administration. A typicalpreparation will contain from about 5% to about 95% active compound orcompounds (w/w). The term “preparation” or “dosage form” is intended toinclude both solid and liquid formulations of the active compound andone skilled in the art will appreciate that an active ingredient canexist in different preparations depending on the desired dose andpharmacokinetic parameters.

The term “excipient” as used herein refers to a compound that is used toprepare a pharmaceutical composition, and is generally safe, non-toxicand neither biologically nor otherwise undesirable, and includesexcipients that are acceptable for veterinary use as well as humanpharmaceutical use. The compounds of this invention can be administeredalone but will generally be administered in admixture with one or moresuitable pharmaceutical excipients, diluents or carriers selected withregard to the intended route of administration and standardpharmaceutical practice.

A “pharmaceutically acceptable salt” form of an active ingredient mayalso initially confer a desirable pharmacokinetic property on the activeingredient which were absent in the non-salt form, and may evenpositively affect the pharmacodynamics of the active ingredient withrespect to its therapeutic activity in the body. The phrase“pharmaceutically acceptable salt” of a compound as used herein means asalt that is pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as glycolic acid, pyruvicacid, lactic acid, malonic acid, malic acid, maleic acid, fumaric acid,tartaric acid, citric acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid, lauryl sulfuric acid, gluconic acid, glutamic acid, salicylicacid, muconic acid, and the like or (2) basic addition salts formed withthe conjugate bases of any of the inorganic acids listed above, whereinthe conjugate bases comprise a cationic component selected from amongNa⁺, K⁺, Mg²⁺, Ca²⁺, NH_(g)R″′_(4-g) ⁺, in which R′″ is a C₁₋₃ alkyl andg is a number selected from among 0, 1, 2, 3, or 4. It should beunderstood that all references to pharmaceutically acceptable saltsinclude solvent addition forms (solvates) or crystal forms (polymorphs)as defined herein, of the same acid addition salt.

Solid form preparations include, for example, powders, tablets, pills,capsules, suppositories, and dispersible granules. A solid carrier maybe one or more substances which may also act as diluents, flavoringagents, solubilizers, lubricants, suspending agents, binders,preservatives, tablet disintegrating agents, or an encapsulatingmaterial. In powders, the carrier generally is a finely divided solidwhich is a mixture with the finely divided active component. In tablets,the active component generally is mixed with the carrier having thenecessary binding capacity in suitable proportions and compacted in theshape and size desired. Suitable carriers include but are not limited tomagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.Solid form preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like. Examples of solid formulations are exemplified in EP 0524579;U.S. Pat. No. 6,635,278; US 2007/0099902; U.S. Pat. No. 7,060,294; US2006/0188570; US 2007/0077295; US 2004/0224917; U.S. Pat. No. 7,462,608;US 2006/0057196; U.S. Pat. No. 6,267,985; U.S. Pat. No. 6,294,192; U.S.Pat. No. 6,569,463; U.S. Pat. No. 6,923,988; US 2006/0034937; U.S. Pat.No. 6,383,471; U.S. Pat. No. 6,395,300; U.S. Pat. No. 6,645,528; U.S.Pat. No. 6,932,983; US 2002/0142050; US 2005/0048116; US 2005/0058710;US 2007/0026073; US 2007/0059360; and US 2008/0014228, each of which isincorporated by reference.

Liquid formulations also are suitable for oral administration includeliquid formulation including emulsions, syrups, elixirs and aqueoussuspensions. These include solid form preparations which are intended tobe converted to liquid form preparations shortly before use. Emulsionsmay be prepared in solutions, for example, in aqueous propylene glycolsolutions or may contain emulsifying agents such as lecithin, sorbitanmonooleate, or acacia. Aqueous suspensions can be prepared by dispersingthe finely divided active component in water with viscous material, suchas natural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well known suspending agents.

The compounds of the present invention may be formulated foradministration as suppositories. A low melting wax, such as a mixture offatty acid glycerides or cocoa butter is first melted and the activecomponent is dispersed homogeneously, for example, by stirring. Themolten homogeneous mixture is then poured into convenient sized molds,allowed to cool, and to solidify.

The compounds of the present invention may be formulated for vaginaladministration. Pessaries, tampons, creams, gels, pastes, foams orsprays containing in addition to the active ingredient such carriers asare known in the art to be appropriate.

Suitable formulations along with pharmaceutical carriers, diluents andexcipients are described in Remington: The Science and Practice ofPharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19thedition, Easton, Pa., which is hereby incorporated by reference. Askilled formulation scientist may modify the formulations within theteachings of the specification to provide numerous formulations for aparticular route of administration without rendering the compositions ofthe present invention unstable or compromising their therapeuticactivity.

The modification of the present compounds to render them more soluble inwater or other vehicle, for example, may be easily accomplished by minormodifications (e.g., salt formulation), which are well within theordinary skill in the art. It is also well within the ordinary skill ofthe art to modify the route of administration and dosage regimen of aparticular compound in order to manage the pharmacokinetics of thepresent compounds for maximum beneficial effect in patients.

Additionally, the purified compounds of the present invention may beformulated in conjunction with liposomes or micelles. As to liposomes,it is contemplated that the purified compounds can be formulated in amanner as disclosed in U.S. Pat. No. 5,013,556; U.S. Pat. Nos.5,213,804; 5,225,212; 5,891,468; 6,224,903; 6,180,134; 5,192,549;5,316,771; 4,797,285; 5,376,380; 6,060,080; 6,132,763; 6,653,455;6,680,068; 7,060,689; 7,070,801; 5,077,057; 5,277,914; 5,549,910;5,567,434; 5,077,056; 5,154,930; 5,736,155; 5,827,533; 5,882,679;6,143,321; 6,200,598; 6,296,870; 6,726,925; and 6,214,375, each of whichis incorporated by reference. As to micelles, it is contemplated thatthe purified compounds can be formulated in a manner as disclosed inU.S. Pat. Nos. 5,145,684 and 5,091,188, both of which are incorporatedby reference.

A sixth embodiment of the present invention is directed to a use of thecompound represented by formula I in the manufacture of a medicament forthe treatment of any condition the result of an infection by any one ofthe following viral agents: hepatitis C virus, West Nile virus, yellowfever virus, dengue virus, rhinovirus, polio virus, hepatitis A virus,bovine viral diarrhea virus and Japanese encephalitis virus.

The term “medicament” means a substance used in a method of treatmentand/or prophylaxis of a subject in need thereof, wherein the substanceincludes, but is not limited to, a composition, a formulation, a dosageform, and the like, comprising the compound of formula I. It iscontemplated that the use of the compound represented by formula I inthe manufacture of a medicament, for the treatment of any of theantiviral conditions disclosed herein, either alone or in combinationwith another compound of the present invention. A medicament includes,but is not limited to, any one of the compositions contemplated by theseventh embodiment of the present invention.

A seventh embodiment of the present invention is directed to a method oftreatment and/or prophylaxis in a subject in need thereof said methodcomprises administering a therapeutically effective amount of thecompound represented by formula I to the subject.

A first aspect of the seventh embodiment is directed to a method oftreatment and/or prophylaxis in a subject in need thereof said methodcomprises administering a therapeutically effective of at least twocompounds falling within the scope of the compound represented byformula I to the subject.

A second aspect of the seventh embodiment is directed to a method oftreatment and/or prophylaxis in a subject in need thereof said methodcomprises alternatively or concurrently administering a therapeuticallyeffective of at least two compounds falling within the scope of thecompound represented by formula I to the subject.

It is intended that a subject in need thereof is one that has anycondition the result of an infection by any of the viral agentsdisclosed herein, which includes, but is not limited to, hepatitis Cvirus, West Nile virus, yellow fever virus, dengue virus, rhinovirus,polio virus, hepatitis A virus, bovine viral diarrhea virus or Japaneseencephalitis virus, flaviviridae viruses or pestiviruses orhepaciviruses or a viral agent causing symptoms equivalent or comparableto any of the above-listed viruses.

The term “subject” means a mammal, which includes, but is not limitedto, cattle, pigs, sheep, chicken, turkey, buffalo, llama, ostrich, dogs,cats, and humans, preferably the subject is a human. It is contemplatedthat in the method of treating a subject thereof of the ninth embodimentcan be any of the compounds contemplated herein, either alone or incombination with another compound of the present invention.

The term “therapeutically effective amount” as used herein means anamount required to reduce symptoms of the disease in an individual. Thedose will be adjusted to the individual requirements in each particularcase. That dosage can vary within wide limits depending upon numerousfactors such as the severity of the disease to be treated, the age andgeneral health condition of the patient, other medicaments with whichthe patient is being treated, the route and form of administration andthe preferences and experience of the medical practitioner involved. Fororal administration, a daily dosage of between about 0.001 and about 10g, including all values in between, such as 0.001, 0.0025, 0.005,0.0075, 0.01, 0.025, 0.050, 0.075, 0.1, 0.125, 0.150, 0.175, 0.2, 0.25,0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, and 9.5, per day should be appropriate in monotherapy and/or incombination therapy. A particular daily dosage is between about 0.01 andabout 1 g per day, including all incremental values of 0.01 g (i.e., 10mg) in between, a preferred daily dosage about 0.01 and about 0.8 g perday, more preferably about 0.01 and about 0.6 g per day, and mostpreferably about 0.01 and about 0.25 g per day, each of which includingall incremental values of 0.01 g in between. Generally, treatment isinitiated with a large initial “loading dose” to rapidly reduce oreliminate the virus following by a decreasing the dose to a levelsufficient to prevent resurgence of the infection. One of ordinary skillin treating diseases described herein will be able, without undueexperimentation and in reliance on personal knowledge, experience andthe disclosures of this application, to ascertain a therapeuticallyeffective amount of the compounds of the present invention for a givendisease and patient.

Therapeutic efficacy can be ascertained from tests of liver functionincluding, but not limited to protein levels such as serum proteins(e.g., albumin, clotting factors, alkaline phosphatase,aminotransferases (e.g., alanine transaminase, aspartate transaminase),5′-nucleosidase, γ-glutaminyltranspeptidase, etc.), synthesis ofbilirubin, synthesis of cholesterol, and synthesis of bile acids; aliver metabolic function, including, but not limited to, carbohydratemetabolism, amino acid and ammonia metabolism. Alternatively thetherapeutic effectiveness may be monitored by measuring HCV-RNA. Theresults of these tests will allow the dose to be optimized.

A third aspect of the seventh embodiment, is directed to a method oftreatment and/or prophylaxis in a subject in need thereof said methodcomprises administering to the subject a therapeutically effectiveamount of a compound represented by formula I and a therapeuticallyeffective amount of another antiviral agent; wherein the administrationis concurrent or alternative. It is understood that the time betweenalternative administration can range between 1-24 hours, which includesany sub-range in between including, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 hours. Examples of“another antiviral agent” include, but are not limited to: HCV NS3protease inhibitors (see WO 2008010921, WO 2008010921, EP 1881001, WO2007015824, WO 2007014925, WO 2007014926, WO 2007014921, WO 2007014920,WO 2007014922, US 2005267018, WO 2005095403, WO 2005037214, WO2004094452, US 2003187018, WO 200364456, WO 2005028502, and WO2003006490); HCV NS5B Inhibitors (see US 2007275947, US20072759300,WO2007095269, WO 2007092000, WO 2007076034, WO 200702602, US 2005-98125,WO 2006093801, US 2006166964, WO 2006065590, WO 2006065335, US2006040927, US 2006040890, WO 2006020082, WO 2006012078, WO 2005123087,US 2005154056, US 2004229840, WO 2004065367, WO 2004003138, WO2004002977, WO 2004002944, WO 2004002940, WO 2004000858, WO 2003105770,WO 2003010141, WO 2002057425, WO 2002057287, WO 2005021568, WO2004041201, US 20060293306, US 20060194749, US 20060241064, US 6784166,WO 2007088148, WO 2007039142, WO 2005103045, WO 2007039145, WO2004096210, and WO 2003037895); HCV NS4 Inhibitors (see WO 2007070556and WO 2005067900); HCV NS5a Inhibitors (see US 2006276511, WO2006120252, WO 2006120251, WO 2006100310, WO 2006035061); Toll-likereceptor agonists (seeWO 2007093901); and other inhibitors (see WO2004035571, WO 2004014852, WO 2004014313, WO 2004009020, WO 2003101993,WO 2000006529); and compounds disclosed in U.S. patent application Ser.No. 12/053,015, filed Mar. 21, 2008 (the contents of which areincorporated by reference).

A fourth aspect of the seventh embodiment, is directed to a method oftreatment and/or prophylaxis in a subject in need thereof said methodcomprises alternatively or concurrently administering a therapeuticallyeffective amount of a compound represented by formula I and anotherantiviral agent to the subject. It is understood that the time betweenalternative administration can range between 1-24 hours, which includesany sub-range in between including, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 hours.

It is contemplated that the another antiviral agent such asinterferon-α, interferon-β, pegylated interferon-α, ribavirin,levovirin, viramidine, another nucleoside HCV polymerase inhibitor, aHCV non-nucleoside polymerase inhibitor, a HCV protease inhibitor, a HCVhelicase inhibitor or a HCV fusion inhibitor. When the active compoundor its derivative or salt are administered in combination with anotherantiviral agent the activity may be increased over the parent compound.When the treatment is combination therapy, such administration may beconcurrent or sequential with respect to that of the nucleosidederivatives. “Concurrent administration” as used herein thus includesadministration of the agents at the same time or at different times.Administration of two or more agents at the same time can be achieved bya single formulation containing two or more active ingredients or bysubstantially simultaneous administration of two or more dosage formswith a single active agent.

It should be noted that for the compounds disclosed herein metabolitesmay be produced upon administration to a subject in need thereof. Forinstance for a compound represented by formula I, it is contemplatedthat hydrolysis of phosphate ester (—OR¹) or the carbonyl ester (−OR⁴)may occur; and that the resultant hydrolyzed product can itself undergoin vivo hydrolysis to form a monophosphate, which can be converted to adiphosphate and/or triphosphate. It is contemplated that the claimsprovided below embrace both synthetic compounds and compounds producedin vivo. The metabolite compounds of the compounds represented bystructures 11-14 (see below) can be obtained by administering saidcompounds to a patient in need thereof. Alternatively, the metabolitecompounds can be prepared by selective hydrolysis of the carbonyl ester(−OR⁴) or the phosphate ester (—OR¹) for compounds represented bystructures 11-14 (see below). As metabolites or metabolite salts arecontemplated herein, it is also contemplated as an alternativeembodiment a method of treatment in a patient in need thereof contactingat least one compound represented by formula I with at least onehepatitis C virus infected cell.

It will be understood that references herein to treatment extend toprophylaxis as well as to the treatment of existing conditions.Furthermore, the term “treatment” of a HCV infection, as used herein,also includes treatment or prophylaxis of a disease or a conditionassociated with or mediated by HCV infection, or the clinical symptomsthereof.

EXAMPLES

A further understanding of the disclosed embodiments will be appreciatedby consideration of the following examples, which are only meant to beillustrative, and not limit the disclosed invention.

The use of the convergent glycosylation route to prepare2′-deoxy-2′-fluoro-2′-C-methyl purine nucleosides and theircorresponding nucleotide phosphoramidates came about with thedevelopment of the synthesis of3,5-di-O-benzoyl-2-deoxy-2-fluoro-2-C-methylribonolactone (Chun, K.;Wang, P. Intl. Pat. Appl. WO 2006/031725).

After several attempts using Vorbrueggen-type Lewis acid mediatedcoupling and the ribonolactol 1-O-acetate of3,5-di-O-benzoyl-2-deoxy-2-fluoro-2-C-methylribonolactone, we observedvery low coupling yields and the undesired α-anomer was the majorproduct. Mitsunobu coupling with the ribonolactol (2) did give thedesired product but with no stereoselectivity and very difficultchromatographic separation resulting in isolated yields of 6-10% forthis step alone and the method was not scaleable.

The preferred approach became the S_(N)2 type reaction using ahalo-sugar and a salt of the purine base. Again, the challenge of thisapproach was how to obtain a a halo-sugar stereospecifically in highyield to take advantage of the inversion of configuration expected withS_(N)2 type reactions. A typical method treats an anomeric mixture ofthe 1-O-acetate of a sugar with HCl or HBr in acetic acid. However, thismethod resulted in production of unfavorable anomeric mixtures. Reducingthe lactone (e.g., with LiAlH(t-BuO)₃ or Red-Al) initially generates at2:1 ratio of β/α anomers but after initial purification through a silicagel filtration column, the resulting oil slowly anomerizes to form purecrystalline β-anomer of the lactol (2). This can be accelerated fromseveral days at ambient temperature to 5-17 h at 50° C. with seedingβ-crystals. We observed that once the lactol is in solution, it slowlyanomerizes back towards the 2:1 equilibrium in solvents such asdichloromethane or chloroform at ambient temperature. This process canbe slowed considerably by chilling the solution (eg −20° C.).

Chlorination through an S_(N)2 mechanism with N-chlorosuccinimide (NCS)produced an α-chlorosugar (3) in a stereospecific manner in almostquantitative yield.

To obtain an α-bromosugar (4), many bromination conditions were triedincluding N-bromosuccinimide (NBS) and HBr in acetic acid. Among them,we followed a general bromination reaction using a combination oftriphenylphosphine (PPh₃) and carbon tetrabromide (CBr₄) (eg. Hooz etal, Can. J. Chem., 1968, 46, 86-87). Under the conditions of usingmethylene chloride as the solvent and maintaining a low temperature (−10to −20° C.) we obtained the best result where the desired α/β isomerratio was greater than 10:1, in a yield of greater than 80%. Applicantsbelieve that there are no literature precedents describing this level ofstereoselectivity for this reaction type. Another practical observationwas that by conducting the bromination under sub-ambient temperatureconditions, such as, most preferably about −20° C.) and exposing thecold reaction solution to silica gel as soon as possible after thecompletion of the reaction minimizes anomerization of the bromosugar.The bromosugar can be purified through a silica gel filtration column.Once treated with silica gel, the bromosugar is practically stable evenat elevated temperatures.

The iodosugar (5) was prepared in a similar manner, which can be coupledwith the purine to produce the key intermediate (6).

Following the general purine coupling method of Bauta et al (Intl. Pat.Appl. WO 2003/011877), we coupled the α-bromosugar (4) with thepotassium salt of 6-chloro-2-amino-purine in t-butanol in acetonitrile.The reaction took over a week at ambient temperatures. The reaction wasoptimized to go to completion in 24 h at 50° C. After partialpurification through a silica gel filtration column, the anomericmixture was isolated in 63% yield in a ratio of 14:1 β/α. The β-anomer(6) could be selectively crystallized out from a methanolic solution togive the pure desired β-anomer (6) in 55% yield from the bromosugar (4).

With the key intermediate 6 in hand, conversion to unprotected2-amino-6-substituted purines (e.g., 7-10) was accomplished. Furtherconversion to the phosphoramidate derivatives (e.g., 11-14) proceeded byan adaptation of the method of Lehsten et al., Org. Proc. Res. Dev.,2002, 6, 819-822 or as disclosed in U.S. patent application Ser. No.12/053,015, filed Mar. 21, 2008, pp. 651-675. As the phosphoramidategroup can also react to a minor extent on the secondary 3′ hydroxyl, thepotential exists for 3′ monophosphoramidate and 3′, 5′bis-phosphoramidate impurities. The 3′ isomer would be expected to havesimilar physical properties to the desired 5′ isomer making purificationby chromatography difficult. This is ameliorated by further reacting thecrude product mixture with sub-stoichiometric amounts of protectinggroups which are selective for primary hydroxyls over secondaryhydroxyls such as t-butyldimethylsilyl chloride, t-butyldiphenylsilylchloride or 4,4′-dimethoxytrityl chloride in the presence of pyridine orsimilar base to generate 5′ protected 3′ phosphoramidate. The resultingproduct and the bis substituted phosphoramidate are less polar than thedesired 5′ phosphoramidate and can be separated readily bychromatography.

Compound (I) can be obtained by a process disclosed at page 5 in U.S.Published Application No. 2008/0139802 (which corresponds to WO2008/045419), at pages 11-13 in WO 2006/012440, and at pages 20-22 and30-31 in WO 2006/031725, each of which is hereby incorporated byreference.

Synthesis of((2R,3R,4R,5R)-3-(benzoyloxy)-4-fluoro-5-hydroxy-4-methyltetrahydrofuran-2-yl)methylbenzoate (2)

To a 5 L of dry three-neck round-bottomed flask fit with a mechanicalstirrer, addition funnel and thermometer was charged the lactone((2R,3R,4R)-3-(benzoyloxy)-4-fluoro-4-methyl-5-oxotetrahydrofuran-2-yl)methylbenzoate) (1, 379 g, 1.018 mol). The solid was dissolved in anhydrousTHF (1.75 L) and cooled to −30° C. under a nitrogen atmosphere. Asolution of lithium tri-tert-butoxyaluminohydride (1.0 M in THF, 1.527L) was added to the lactone solution while stirring over 1 h andmaintaining the −30° C. temperature. After finishing the addition, thetemperature was slowly increased and the reaction was followed by TLC(lactol R_(f) 0.4, 30% EtOAc in hexanes). The reaction was completeafter 1 h 15 min (temperature reached −10° C.). The reaction wasquenched by addition of Ethyl acetate (900 mL) via addition funnel Sat.NH₄Cl (40 mL) was added at 0° C. The cloudy mixture was decanted into a10 L round-bottomed flask. The solid residue left behind was filteredand washed with ethyl acetate (2×200 mL). The filtrate was combined withthe decanted solution and the combined solution was concentrated underreduced pressure. The oily residue was dissolved in ethyl acetate (2 L)and washed with 3 N HCl (600 mL). The aqueous layer was back-extractedwith ethyl acetate (3×400 mL). The combined organic layer was washedwith water (3×800 mL), sat. NaHCO₃ (400 mL) and brine (400 mL). Theorganic solution was dried over MgSO₄, filtered and concentrated underreduced pressure to afford a light brown oily residue. The residue waspurified by plug column (2.2 kg of 40-63 micron silica gel, packed in a6 L sintered glass funnel, 22 cm length of silica gel, diameter 15 cm)using suction and a step-gradient of 5%, 10%, 20%, and 30% ethyl acetatein hexanes ca 5 L of each). The product containing fractions werecombined and concentrated under reduced pressure to a colorless, verythick liquid (310.4 g).

The liquid slowly solidified after adding crystalline beta product asseeds (ca 100 mg spread out) under vacuum (0.2 mmHg) at 50° C. Theprocess of solidification was complete in 20 hours at 50° C. with orwithout vacuum. The white solid thus collected (293.8 g, 77%) has amp of79-80° C. and ratio of β/α is 20:1 based on NMR.

¹H-NMR (DMSO-d₆) 13-isomer, δ=5.20 (dd, 1H, OH); α-isomer, δ=5.40 (dd,1H, OH). (β-lactol). (DMSO-d₆): δ 7.99 (m, 2H, arom.), 7.93 (m, 2H,arom.), 7.70 (m, 1H, arom.), 7.61 (m, 1H, arom.), 7.55 (m, 2H, arom.),7.42 (m, 2H, arom.), 7.32 (dd, 1H, C1-H), 5.54 (dd, 1H, C3-H), 5.20 (dd,1H, OH), 4.55-4.50 (m, 1H, C5-Ha), 4.46-4.40 (m, 2H, C5-Hb) and C4-H),1.42 (d, 3H, CH₃).

Synthesis of((2R,3R,4R,5R)-3-(benzoyloxy)-5-chloro-4-fluoro-4-methyltetrahydrofuran-2-yl)methylbenzoate (3)

To a solution of mixture of compound 2 (1.0 g, 2.67 mmol) and PPh₃ (1.4g, 5.34 mmol) in CH₂Cl₂ (15 mL) was added NCS (1.07 g, 8.01 mmol)portionwise at 0° C. Then the resulting mixture was stirred at rt for 1h and poured into a silica gel column and eluted with EtOAc-hexanes(1:4) using pressure. The collected right fractions were combined,concentrated, and co-evaporated with CH₂Cl₂ several times and used nextstep (1.0 g, 95%).

¹H-NMR (CDCl₃) δ=8.13-8.02 (m, 4H, aromatic), 7.78-7.50 (m, aromatic,2H), 7.53-7.43 (m, 4H, aromatic), 6.01 (s, 1H, H-1), 5.28 (dd, 1H,J=3.2, 5.6 Hz, H-3), 4.88 (m, 1H, H—H-4), 4.77 (dd, 1H, J=3.2, 12.4 Hz,H-5), 4.61 (dd, 1H, J=4.0, 12.4 Hz, H-5′), 1.73 (d, 3H, J=21.6 Hz, CH₃).

Synthesis of((2R,3R,4R,5R)-3-(benzoyloxy)-5-bromo-4-fluoro-4-methyltetrahydrofuran-2-yl)methylbenzoate (4)

Anhydrous dichloromethane (5.6 L) was charged into a reactor and cooledto −22° C. or below. Triphenylphosphine (205.4 g, 0.783 mol) was addedto the cold solvent and the suspension was stirred to form a solution.The lactol (2, 209.4 g, 0.559 mol) in solid form was added to the coldsolution and stirred for 15 mins. Carbon tetrabromide (278.2 g, 0.839mol) was added portion-wise while maintaining the temperature of thesolution between −22° C. to −20° C. under a flow of nitrogen gas(approx. 30 min). After finishing the addition of CBr₄, the temperaturewas slowly raised to −17° C. over 20 mins. The reaction was judged tobe >95% complete by TLC (R_(f)s 0.61 (α), 0.72 ((β), 0.36 lactol; 20%EtOAc in hexanes). The reaction solution was immediately transferred toa vessel containing 230 g of flash chromatography grade silica gel(40-63 microns). The stirred mixture was immediately passed through apad of silica gel (680 g) in a 2.5 L sintered glass Buchner funnel. Thefiltrate was concentrated under reduced pressure to about 800 mL and theratio of α/β isomers of the crude product was 10:1 as determined by¹H-NMR. (CDCl₃) δ=6.35, (s, α C1-H), 6.43, (d, β C1-H). The residue waspurified by plug column chromatography using 2.1 kg of silica gel in a 6L sintered glass Buchner funnel and eluted (via suction) with a stepwisegradient elution of 1%, 5%, 8% 12% EtOAc in hexane (ca 4 L each) toremove non-polar impurities followed by 12%, 25% EtOAc in hexane (6 Ltotal) to elute the product. The product containing fractions werecombined into two fractions, concentrated under reduced pressure, driedunder vacuum (0.1 mmHg, ambient temp., 20 h) to colorless oils. Mainfraction (197 g, 89% α/β=20:1). The alpha isomer crystallized from asmall portion of the oil upon standing at 0° C. for several weeks togive large, thin plates, mp 59-61° C. The pure beta isomer crystallizedfrom a mixture of alpha and beta product oil from an earlier lessselective run to give needles, mp 77-79° C.

¹H-NMR (β-bromide) (CDCl₃): δ=8.08 (m, 2H, arom.), 8.04 (m, 2H, arom.),7.62 (m, 1H, arom.), 7.54-7.45 (m, 3H, arom.), 7.35 (m, 2H, arom.), 6.43(d, 1H, C1-H), 6.04 (dd, 1H, C3-H), 4.78-4.73 (m, 2H, C4-H and C5-Ha),4.63-4.58 (m, 1H, C5-Hb), 1.76 (d, 3H, CH₃). α-bromide, α/β=20:1)(CDCl₃): δ 8.13 (m, 2H, arom.), 8.02 (m, 2H, arom.), 7.63-7.56 (m, 2H,arom.), 7.50-7.42 (m, 4H, arom.), 6.34 (s, 1H, C1-H), 5.29 (dd, 1H,C3-H), 4.88 (m, 1H, C4-H), 4.78 (dd, 1H, C5-Ha), 4.63 (dd, 1H, C5-Hb),1.72 (d, 3H, CH₃).

Synthesis of((2R,3R,4R,5R)-3-(benzoyloxy)-4-fluoro-5-iodo-4-methyltetrahydrofuran-2-yl)methylbenzoate (5)

To a solution of compound 2 (1 g, 2.67 mmol), triphenylphosphine (700mg, 2.67 mmol), and imidazole (180 mg, 2.67 mmol) in anhydrous CH₂Cl₂(10 mL) iodine (680 mg, 2.68 mmol) was added. The resulting mixture wasstirred for 30 min and poured into a silica gel column and eluted withEtOAc-hexanes (1:4) to give a syrupy product (1.3 g, quantitative) andused in next reaction without further characterization.

Synthesis of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate (6)

To a 12 L of three-neck round-bottomed flask was charged6-chloro-2-aminopurine (225.4 g, 1.329 mol). Anhydrous tert-BuOH (4.5 L)was added and the solution was stirred with a mechanical stirrer atambient temperature. Potassium tert-butoxide (solid, 151.6 g, 1.35 mol)was added portion-wise under a flow of nitrogen gas while stirring. Themixture was stirred at RT for an additional 30 min. To a 5 Lround-bottomed flask was loaded the α-bromide (4, 197 g, 0.451 mol) and3 L of anhydrous acetonitrile at ambient temperature. The bromidesolution was added to the purine base suspension over 1 min at ambienttemperature. The 5 L flask was rinsed with acetonitrile (2×1 L) totransfer bromide completely to the reaction mixture. The mixture washeated gradually to 50° C. over 2 h with a heating mantle andcontroller, and stirred for 20 h. The reaction was almost complete asshown by TLC beta (R_(f) 0.28, 30% EtOAc in hexanes). The reaction wasquenched by the addition of sat. NH₄Cl (200 mL) to form a suspension.The suspended solid¹ was removed by filtration through a 3 cm pad ofCelite in a 2.5 L porcelain Buchner funnel. The solid was washed withtoluene (3×100 mL). The combined filtrate was neutralized by adding 6 NHCl solution until pH 7 (approx 220 mL). The mixture was concentratedunder reduced pressure. When the volume of mixture was reduced to aboutone-third volume, additional precipitated solid was removed byfiltration in a similar manner. The filtrate was further concentrated toa volume of about 800 mL. The residue was loaded onto a plug column (1.6kg flash grade silica gel in a 6 L sintered glass Buchner funnel) andeluted (via suction) with a gradient of 10% ethyl acetate in hexanes (6L) to remove non-polar impurities, 30% ethyl acetate in hexanes toafford a small amount of lactol (6 L), and then 40%-45% ethyl acetate inhexanes (4 L) to elute the main amount of product. The productcontaining fractions were combined, concentrated under reduced pressureand dried under vacuum (0.2 mmHg, 24 h, ambient temp.) to a white foamsolid (150.7 g, α/β=14:1 by NMR.

¹H-NMR. (CDCl₃) beta: δ=1.33 (d, 22.4 Hz, 2′-C—CH₃), alpha: 1.55 (d, 22Hz, 2′-C—CH₃).

The product mixture foam was dissolved in methanol (700 mL) at ambienttemperature. Upon standing, a solid slowly formed over 2 h. Thesuspension was cooled in a freezer to −5° C. for 17 h. The resultingwhite solid was collected by filtration and washed with cold MeOH (−5°C., 3×60 mL) and ethyl ether (3×100 mL). The solid was dried undervacuum (0.2 mmHg, 24 h, ambient temp.) to afford 110.5 g of β-productwith excellent de α/β 99.8:1 by HPLC). The filtrate was partiallyconcentrated (ca. 400 mL) and then diluted with more MeOH (400 mL) whileheating to 60° C. The solution was cooled down to ambient temperature,seeded and then cooled to −5° C. The second crop was collected, washedand dried in a similar manner to give more product as a white solid(12.26 g) with similar diastereomeric purity. The mother liquor wasconcentrated to dryness under reduced pressure (ca. 25 g). The residuewas a mixture of p and α-isomers. It was subjected to automated silicagel column chromatography (Analogix, 240 g cartridge, 40% to 50% ethylacetate in hexanes) to afford 14.52 g of product foam which wasrecrystallized from MeOH, washed and dried in a similar manner to affordan additional 8.46 g of product in high purity.

The three solids were judged to be of similar purity and they werecombined to give 131.2 g of white crystalline product 6, (55% frombromosugar, 49% from lactol). Mp 160.5-162.0° C. HPLC purity 99.5%including 0.20% alpha.

¹H-NMR (pure β-anomer, CDCl₃): δ=8.03 (m, 2H, arom.), 7.93 (m, 2H,arom.), 7.88 (s, 1H, C8-H), 7.60 (m, 1H, arom.), 7.50 (m, 1H, arom.),7.44 (m, 2H, arom.), 7.33 (m, 2H, arom.), 6.44 (dd, 1H, C1′-H), 6.12 (d,1H, C3′-H), 5.35 (s, 2H, NH₂), 5.00 (dd, 1H, C5′-Ha), 4.76 (m, 1H,C4′-H), 4.59 (dd, 1H, C5′-Hb), 1.33 (d, 3H, CH₃).

¹H-NMR (α-isomer, CDCl₃): δ=8.11-8.09 (m, 3H, arom. and C8-H), 8.01 (m,2H, arom.), 7.63 (m, 1H, arom.), 7.55 (m, 1H, arom.), 7.48 (m, 2H,arom.), 7.39 (m, 2H, arom.), 6.35 (d, 1H, C1′-H), 5.76 (dd, 1H, C3′-H),5.18 (s, 2H, NH₂), 4.93-4.89 (m, 1H, C4′-H), 4.75-4.71 (m, 1H, C5′-Ha),4.58-4.54 (m, 1H, C5′-Hb), 1.55 (d, 3H, CH₃).

Synthesis of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate (6) from compound 3

To a solution of compound 3 (450 mg, 2.68 mmol) in chlorobenzene (1.5mL) were added potassium salt of the base (1.37 g, 8.05 mmol) int-butanol (5 mL) and subsequently anhydrous acetonitrile (5 mL) at rt.The resulting mixture was stirred at 80-140° C. in a sealed tube for 7days and concentrated in vacuo after neutralization with HCl. Theresidue was purified by silica gel column chromatography(hexanes:EtOAc=2:1) to give compound 6 (90 mg, 15%) as a white foam.

Synthesis of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate (6) from compound 5

To a solution of compound 5 (1.3 g, 2.68 mmol) in t-butanol (10 mL) wasadded sodium salt of the base (1.37 g, 8.05 mmol) in DMF (10 mL) atambient temperature. The resulting mixture was stirred for 15 h andconcentrated in vacuo. The residue was purified by silica gel columnchromatography (hexanes:EtOAc=2:1) to give compound 6 (220 mg, 16%) as awhite foam.

Synthesis of(2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(7)

To a 250 mL dry round-bottomed flask was charged(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate (6, 7.50 g, 14.26 mmol). Anhydrous methanol (30 mL) was addedand a white suspension was formed. At 50° C., a solution of sodiummethoxide in methanol (25%, 19.7 mL, 64.17 mmol) was added via a drysyringe under a nitrogen atmosphere. A white cloudy reaction mixture wasformed. After 3.5 h at 50° C., the reaction was complete with nostarting material left as shown by TLC test. The mixture was cooled downto room temperature and neutralized by addition of glacial acetic acid(3 mL). A white solid was filtered out and washed with methanol (3×5mL). The filtrate was mixed with 20 g of silica gel and concentrated todryness. The mixture was loaded in line with a silica gel cartridge andseparated via column chromatography using a gradient of methanol indichloromethane 0 to 15% MeOH. The product eluted out at 12% methanol indichloromethane. The product containing fractions were combined,concentrated under reduced pressure and dried under vacuum (0.2 mmHg,50° C., 24 h) to a white powder solid (4.45 g, 98% yield), mp 199-202°C.

¹H-NMR (DMSO-d₆): δ=8.18 (1H, s, C8-H), 6.61 (2H, s, NH₂), 6.05 (1H, d,C1′-H), 5.68 (1H, d, 3′-OH), 5.26 (1H, m, 5′-OH), 4.23-4.13 (1H, m,C3′-H), 3.96 (3H, s, OCH₃), 3.92-3.83 (2H, m, C4′-H and C5′-H_(a)),3.70-3.67 (1H, m, C5′-H_(b)), 1.06 (3H, d, C2′-CH₃).

Synthesis of (2S)-isopropyl2-((((2R,3R,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy) phosphorylamino)propanoate (11)

To a 250 mL dry round-bottomed flask were loaded phenyldichlorophosphate (2.66 g, 12.61 mmol) and anhydrous dichloromethane (40mL). The amino ester salt (2.60 g, 15.53 mmol) was added to the solutionand the mixture was cooled to −5° C. N-Methyl imidazole (7.7 mL, 97mmol) was then added quickly via a dry syringe at −5° C. and thesolution was stirred at −5° C. for 1 h. The nucleoside (7, 3.04 g, 9.7mmol) was added from a vial in one portion at −5° C. and the solid wasslowly dissolved in 20 minutes. The reaction temperature was allowed torise to ambient temperature over 2 h. After 17 h, the reaction was notcomplete. More reagents were made (as described above from phosphate(2.66 g), aminoester (2.60 g), and NMI (3.8 mL, 48 mmol)) and added tothe reaction mixture at −5° C. The reaction was stirred at roomtemperature for 2 more hours. The reaction was almost complete as shownby TLC result and diluted with 70 mL of dichloromethane. HCl solution (1N, 70 mL) was added. The aqueous layer was separated and extracted withdichloromethane. The organic layer was washed with saturated NaHCO₃,water, brine and dried over MgSO₄. After removal of the solvent underreduced pressure, the sticky residue was purified through automatedcolumn chromatography using a 240 g cartridge and a gradient of 0-8%2-PrOH in dichloromethane to afford product as a foam solid (4.16 g,7.14 mmol, 73% yield). HPLC purity 97.4%. NMR spectra of product showedit is a mixture of two diastereoisomers with a ratio of 1.2:1.

¹H-NMR (DMSO-d₆): δ=7.98 (1H, s, 8-H of one isomer), 7.95 (1H, s, 8-H ofanother isomer), 7.37-7.32 (2H, m, arom-H), 7.22-7.15 (3H, m, arom-H),6.6 (2H, s, NH₂), 6.11 (1H, d, C1′-H of one isomer), 6.09 (1H, d, C1′-Hof another isomer), 6.09-5.98 (1H, m, amide NH), 5.88 (1H, d, 3′-OH ofone isomer), 5.81 (1 H, d, 3′-H of another isomer), 4.85-4.75 (1H,hepta, methine H of iso-propyl), 4.46-4.27 (2H, m, C4′-H, α-H of aminoester), 4.15-4.07 (1H, m, C3′-H), 3.96 (3H, s, OCH₃), 3.82-3.72 (2H, m,C5′-H_(a) and C5′-Hb), 1.23-1.06 (9H, m, CH₃'s of amino ester), 1.03(3H, d, C2′-CH₃).

³¹P-NMR (DMSO-d₆): δ=4.91 (one isomer), 4.72 (another isomer).

An alternate purification method is to chemically alter the minor 3′phosphoramidate by-product in order to simplify the chromatographicseparation. The crude phosphoramidate product is dissolved in anhydrouspyridine (5 mL/g), and is treated with 0.5 molar equivalents oft-butyldimethylsilyl chloride at ambient temperature to reactselectively with the free 5′ primary hydroxyl of the 3′ isomer impurity.Reaction progress can be monitored by LC/MS. Once the 3′ isomer isconverted to a 5′-tBDMS-3′-phosphoramidate derivative, the reaction isquenched with methanol (3 eq), concentrated under reduced pressure,partitioned between ethyl acetate and 5% citric acid and then theorganic layer is concentrated. The residue is then subjected tochromatography which can now be done with a higher loading and a fastergradient and achieve a higher purity.

Synthesis of(2R,3R,4R,5R)-5-(2-amino-6-(azetidin-1-yl)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(8)

To a 350 mL of dry seal pressure flask (Chemglass) were added(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate (6, 3.6 g, 6.85 mmol) and 150 mL of absolute ethanol. Azetidinehydrochloride (2.56 g, 27.4 mmol) was added and then followed bytriethylamine (4.16 g, 41.1 mmol). The suspension was stirred and heatedto 70° C. while sealed for 5 hours. All the starting material wasconsumed but the benzoyl groups remained as shown by TLC. Sodiummethoxide (7.8 mL, 34.3 mmol, 25% solution in methanol) was added to themixture and heated at 50° C. The reaction was complete after 3.5 h. Thereaction mixture was allowed to cool to room temperature and neutralizedby addition of glacial acetic acid (0.41 g, 6.85 mmol). The mixture wasconcentrated under reduced pressure and then the residue was trituratedwith ethyl acetate. The resulting solid was removed by filtration andthe solid was washed with EtOAc (2×15 mL). The filtrate was concentratedunder reduced pressure and the residue was purified via columnchromatography (Analogix, 120 g cartridge, gradient of 0 to 15% MeOH inDCM). The pure product containing fractions were combined, concentratedunder reduced pressure and dried (50° C., 0.2 mmHg, 17 h) to a lightpink colored foam solid (2.15 g, 6.35 mmol, 93%).

¹H-NMR (DMSO-d₆): δ=8.00 (s, 1H, C8-H), 6.03 (s, 2H, NH₂), 6.00 (d, 1H,C1′-H), 5.64 (d, 1H, 3′-OH), 5.24 (t, 1H, 5′-OH), 4.24-4.10 (m, 5H,N—CH₂ of azetidine, C3′-H), 3.90-3.81 (m, 2H, C4′-H and C5′-H_(a)),3.69-3.64 (m, 1H, C5′-H_(b)), 2.37 (penta, 2H, center CH₂ of azetidine),1.05 (d, 3H, C2′-CH₃).

Synthesis of (2S)-methyl24(((92R,3R,4R,5R)-5-(2-amino-6-(azetidin-1-yl)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate (12)

To a 100 mL dry round-bottomed flask were added phenyl dichlorophosphate(1.72 g, 8.15 mmol) and anhydrous dichloromethane (17 mL). The aminoester (1.42 g, 10.2 mmol) was added and the suspension was cooled to −5°C. N-Methylimidazole (3.34 g, 40.7 mmol) was added via a syringe in oneportion and the solution was stirred at −5° C. for 1 h under a nitrogenatmosphere. The nucleoside (8, 1.38 g, 4.07 mmol) (foam solid) was thenadded in one portion and the solution was allowed to warm up over 1 h toambient temperature. After 4 h at ambient temperature, TLC (5% MeOH inDCM) indicated an incomplete reaction (about 30% SM remained) but also agrowing less polar impurity. The reaction was quenched by the additionof sat NH₄Cl (20 mL) and diluted with dichloromethane (20 mL). Theorganic layer was separated and washed with water (5×30 mL), brine (20mL) and dried over Na₂SO₄. The product containing solution was filteredand concentrated under reduced pressure to a crude oily residue, 3.26 g.This was purified by column chromatography (Analogix, 40 g cartridge,gradient of MeOH in DCM from 0% to 10%). The product eluted at 4% MeOHin DCM. The pure product containing fractions were combined,concentrated under reduced pressure and dried (50° C., 0.2 mmHg, 17 h)to a white foam solid (1.322 g, 2.28 mmol, 56%). HPLC purity 99.25%. NMRspectra of product showed it is a mixture of two diastereoisomers with aratio of 55:45.

¹H-NMR (DMSO-d₆): δ=7.80 (s, 1H, 8-H of one isomer), 7.80 (s, 1H, 8-H ofanother isomer), 7.38-7.33 (m, 2H, arom-H), 7.22-7.14 (m, 3H, arom-H),6.09 (s, 2H, NH₂), 6.12-6.02 (m, 2H, C1-H and NH), 5.83 (d, 1H, 3′-OH ofone isomer), 5.77 (d, 1H, 3′-OH of another isomer), 4.46-4.05 (m, 8H,NCH₂ of azetidine, α-H of aminoester, C3′-H, C4′-H, C5′-H_(a)),3.89-3.79 (m, 1H, C5′-H_(b)), 3.56 (s, 3H, OCH₃ of aminoester in oneisomer), 3.54 (s, 3H, OCH₃ of aminoester in another isomer), 2.37(penta, 2H, center CH₂ of azetidine), 1.21 (d, 3H, α-CH₃ of aminoesterin one isomer), 1.19 (d, 3H, α-CH₃ of aminoester in another isomer),1.08 (d, 3H, C2′-CH₃).

³¹P NMR (DMSO-d₆): δ=4.85 (one isomer), 4.77 (other isomer).

Synthesis of(2R,3R,4R,5R)-5-(2-amino-6-(benzyloxy)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(9)

To a 500 mL of dry round-bottomed flask were added(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate (6, 8.0 g, 15.2 mmol) and anhydrous benzyl alcohol (128 mL). Toanother 250 mL of dry round-bottomed flask were charged NaH (60% inmineral oil, 2.44 g, 60.8 mmol) and anhydrous DMF (40 nth). Thesuspension was stirred at 0° C. in an ice-water bath. Benzyl alcohol (27mL) was added drop-wise via a syringe. A solution was slowly formed andit was transferred to the nucleoside suspension quickly under a nitrogenatmosphere at room temperature. The mixture was heated to 50° C. andstirred. The reaction was complete after 3 h and cooled to ambienttemperature. It was neutralized by the addition of 4 N HCl to ca. pH=7(12 mL). The solution was concentrated under reduced pressure (4 mbar,90° C. bath). The cloudy residue was diluted with dichloromethane (100mL) and washed with water (3×30 mL), brine (30 mL) and dried overNa₂SO₄. The suspension was filtered and the filtrate was concentratedunder reduced pressure to an oily residue. This was purified by columnchromatography (Analogix, 0 to 8% gradient of MeOH in DCM). The producteluted at 4% MeOH in DCM. The product containing fractions werecombined, concentrated under reduced pressure and dried (50° C., 0.2mmHg, 17 h) to a white foam solid (4.57 g, 11.7 mmol, 77.2%).

¹H-NMR (DMSO-d₆) δ=8.18 (s, 1H, 8-H), 7.53-7.51 (m, 2H, arom-H),7.43-7.34 (m, 3H, arom-H), 6.66 (s, 2H, NH₂), 6.05 (d, 1H, 5.67 (d, 1H,3′-OH), 5.48 (dd, 2H, CH₂ of Benzyl), 5.25 (t, 1H, 5′-OH), 4.18 (dt, 1H,C3′-H), 3.92-3.82 (m, 2H, C4′-H and C5′-H₄), 3.71-3.66 (m, 1H,C5′-H_(b)), 1.07 (d, 3H, C2′-CH₃).

Synthesis of (2S)-cyclopentyl2-((((2R,3R,4R,5R)-5-(2-amino-6-(benzyloxy)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate (13)

To a 100 mL of dry round-bottomed flask were charged phenyldichlorophosphate (3.29 g, 15.58 mmol) and anhydrous dichloromethane (24mL). The aminoester tosylate (white powder) was added and the solutionwas cooled to −5° C. under nitrogen. N-Methylimidazole (4.92 g, 59.94mmol) was added via a dry syringe in one portion and the resultedcolorless clear solution was stirred at −5° C. for one hour. Then thenucleoside (9) solid was added (2.334 g, 5.99 mmol) to the solutionunder nitrogen in one portion and the mixture was allowed to warm toambient temperature to give a colorless solution. Reaction progress wasmonitored by TLC (5% methanol in dichloromethane). TLC indicated anincomplete reaction after 20 h (about 30% starting material left). Thereaction was still quenched by the addition of dichloromethane (30 mL)and 1 N HCl (60 mL). The organic layer was separated and the aqueouslayer was extracted with dichloromethane (2×20 mL). The combined organiclayer was washed with water (2×40 mL), sat NaHCO₃ (30 mL), water, andbrine. The organic layer was dried over Na₂SO₄. After removal of solidby filtration, the filtrate was concentrated under reduced pressure to agummy residue (7.28 g). The residue was purified via columnchromatography (Analogix, 80 g cartridge, gradient of 0 to 10% MeOH inDCM). The product eluted at 2% MeOH in DCM. The product containingfractions were combined, concentrated under reduced pressure and dried(50° C., 0.2 mmHg, 17 h) to a white foam solid (2.249 g, a mixture oftwo isomers, 60:40). A portion of the starting nucleoside (0.257 g) wasalso recovered. Yield is 62% based on consumed starting material.

¹H-NMR (DMSO-d₆): δ=7.98 (s, 1H, 8-H of one isomer), 7.96 (s, 1H, 8-H ofanother isomer), 7.52-7.50 (m, 2H, arom-H), 7.42-7.31 (m, 5H, arom-H),7.21-7.12 (m, 3H, arom-H), 6.68 (s, 2H, NH₂), 6.12 (d, 1H, C′—H of oneisomer), 6.10 (d, 1H, C1′-H of another isomer), 6.04-5.96 (m, 1H, NH),5.87 (d, 1H, 3′-OH of one isomer), 5.81 (d, 1H, 3′-OH of anotherisomer), 5.48 (dd, 2H, CH₂ of Benzyl), 4.99-4.93 (m, 1H, α-H ofaminoester), 4.46-4.27 (m, 3H, C3′-H, C4′-H, OCH of aminoester),4.15-4.06 (m, 1H, C5′-H_(a)), 3.81-3.71 (m, 1H, C5′-H_(b)), 1.74-1.43(m, 8H, methylene CH₂ of c-pentyl), 1.18 (d, 3H, α-CH₃ of aminoester),1.09 (d, 3H, C2′-CH₃ of one isomer), 1.08 (d, 3H, C2′-CH₃ of anotherisomer).

³¹P NMR (DMSO-d₆): δ=4.91 (one isomer), 4.73 (other isomer).

Synthesis of (2S)-cyclopentyl2-((((2R,3R,4R,5R)-5-(2-amino-6-hydroxy-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate (14)

To a 250 mL of dry round-bottomed flask with starting material (13, 1.92g, 2.8 mmol) was added anhydrous absolute ethanol (50 mL). Palladium oncharcoal (10%, 120 mg) was added. The atmosphere in the flask wasexchanged with hydrogen and the mixture was stirred under 1 atm ofhydrogen gas for 3.5 h at room temperature. The reaction was judgedcomplete by TLC and the Pd on charcoal was removed by filtration andwashed with ethanol (2×10 mL). The filtrate was concentrated underreduced pressure to a solid residue. The solid was mixed with silica gel(10 g) and purified by column chromatography (Analogix, 40 g cartridge,gradient of 1% to 16% MeOH in DCM). The product containing fractionswere combined, concentrated under reduced pressure and dried (50° C.,0.2 mmHg, 17 h) to a white powder (1.43 g, 86%). HPLC purity 99.55%. NMRspectra of product showed it is a mixture of two diastereoisomers with aratio of 60:40. Mp=133-150° C.

¹H-NMR (DMSO-d₆): δ=10.70 (s, 1H, NH of imide), 7.81 (s, 1H, 8-H of oneisomer), 7.79 (s, 1H, 8-H of another isomer), 7.38-7.33 (m, 2H, arom-H),7.22-7.14 (m, 3H, arom-H), 6.62 (s, 2H, NH₂), 6.08-5.97 (m, 2H, C1′-Hand NH of aminoester), 5.88 (b, 1H, 3′-OH of one isomer), 5.82 (b, 1H,3′-OH of another isomer), 5.01-4.94 (m, 1H, α-H of aminoester),4.44-4.25 (m, 3H, C3′-H, C4′-H, OCH of aminoester), 4.12-4.04 (m, 1H,C5′-H_(a)), 3.82-3.72 (m, 1H, C5′-H_(b)), 1.77-1.46 (m, 8H, methyleneCH₂ of c-pentyl), 1.21-1.19 (m, 3H, α-CH₃ of aminoester), 1.09 (d, 3H,C2′-CH₃ of one isomer), 1.08 (d, 3H, C2′-CH₃ of another isomer).

³¹P-NMR (DMSO-d₆): δ=4.95 (one isomer), 4.72 (another isomer).

Synthesis of(2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(10)

To a 500 mL of dry round-bottomed flask was loaded (6, 11 g, 20.92mmol). Anhydrous absolute ethanol (210 mL) was added and followed byanhydrous K₂CO₃ (28:91 g, 209.2 mmol). The suspension was stirred andheated at 75° C. under nitrogen for 5.5 h. All the starting material wasconsumed at that time by TLC test. The mixture was cooled to roomtemperature and solid was filtered out. The filtrate was neutralized byaddition of glacial acetic acid (2.52 g) to pH-7 and concentrated underreduced pressure. The residue was dissolved in methanol and mixed withsilica gel (15 g). The dried mixture of crude product and silica gel wastransferred to an empty cartridge and separated through columnchromatography (Analogix 220 g, gradient of 0 to 15% MeOH in DCM) toafford product (5% MeOH in DCM) as a white foam solid (3.73 g, 54.5%). Asecond white solid was isolated from column (10% MeOH in DCM, 1.44 g)and it is a mixture of two dimers of nucleoside. A more polar, thirdwhite solid was collected from column (15% MeOH in DCM, 0.47 g) and itis a mixture of trimers of nucleoside. HPLC purity of product 99.94%.

¹H-NMR (DMSO-d₆): δ 8.16 (s, 1H, 8-H), 6.55 (s, 2H, NH₂), 6.04 (d, 1H,C′—H), 5.66 (d, 1H, 3′-OH), 5.24 (m, 1H, 5′-OH), 4.44 (q, 2H, 6-OCH₂),4.23-4.08 (m, 1H, C3′-H), 3.91-3.82 (m, 2H, C4′-H and C5′-H_(a)),3.71-3.66 (m, 1H, C5′-H_(b)), 1.36 (t, 3H, CH₃ of ethyl), 1.06 (d, 3H,C2′-CH₃).

Biological Data

HCV Replicon Assay.

HCV replicon RNA-containing Huh7 cells (clone A cells; Apath, LLC, St.Louis, Mo.) were kept at exponential growth in Dulbecco's modifiedEagle's medium (high glucose) containing 10% fetal bovine serum, 4 mML-glutamine and 1 mM sodium pyruvate, 1× nonessential amino acids, andG418 (1,000 μg/ml). Antiviral assays were performed in the same mediumwithout G418. Cells were seeded in a 96-well plate at 1,500 cells perwell, and test compounds were added immediately after seeding.Incubation time 4 days. At the end of the incubation step, totalcellular RNA was isolated (RNeasy 96 kit; Qiagen). Replicon RNA and aninternal control (TaqMan rRNA control reagents; Applied Biosystems) wereamplified in a single-step multiplex RT-PCR protocol as recommended bythe manufacturer. The HCV primers and probe were designed with PrimerExpress software (Applied Biosystems) and covered highly conserved5′-untranslated region (UTR) sequences (sense primer:5′-AGCCATGGCGTTAGTA(T)GAGTGT-3′ (SEQ ID NO:1); antisense primer:5′-TTCCGCAGACCΔCTATGG-3′ (SEQ ID NO:2); and probe:5′-FAM-CCTCCAGGACCCCCCCTCCC-TAMRA-3′ (SEQ ID NO:3)).

To express the antiviral effectiveness of a compound, the thresholdRT-PCR cycle of the test compound was subtracted from the averagethreshold RT-PCR cycle of the no-drug control (ΔCt_(HCV)). A ΔCt of 3.3equals a 1-log 10 reduction (equal to the 90% effective concentration[EC₉₀]) in replicon RNA levels. The cytotoxicity of the test compoundcould also be expressed by calculating the ΔCT_(RNA) values. The ΔΔCtspecificity parameter could then be introduced (ΔCt_(HCV)-ΔCt_(RNA)), inwhich the levels of HCV RNA are normalized for the rRNA levels andcalibrated against the no-drug control.

Compounds 11, 12, and 14, are represented by the following structure(s),

were tested for their biological properties based on the precedingassay. The results of these tests are disclosed in the Table 1.

TABLE 1 Activity of Selected Compounds CloneA EC₉₀ Compd. No. (μM) 110.02 12 0.07 14 0.13

The contents of U.S. patent application Ser. No. 12/053,015, filed Mar.21, 2008 (see also WO 2008/121634), U.S. patent application Ser. No.12/479,075, filed Jun. 5, 2009, and U.S. Provisional Patent ApplicationNos. 61/060,683, filed Jun. 11, 2008, 61/140,441, 61/140,317, and61/140,369, each of which being filed Dec. 23, 2008 are herebyincorporated by reference in their entirety. Moreover, the patent andnon-patent references disclosed herein are incorporated by reference. Inthe event that the incorporated subject matter contains a term thatconflicts with a term disclosed in the present application text, themeaning of the term contained in the present application controlsprovided that the overall meaning of the incorporated subject matter isnot lost.

1. A compound represented by formula I, or its stereoisomers, salts, or pharmaceutically acceptable salts thereof:

wherein Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ is hydrogen, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; R⁸ is OMe, —N(—CH₂CH₂CH₂—), OBn, or OH; and R⁹ is NH₂.
 2. A pharmaceutical composition comprising a pharmaceutically acceptable medium and the compound according to claim
 1. 3. A method of treatment of a subject infected with a virus selected from the group consisting of hepatitis C virus, West Nile virus, yellow fever virus, dengue virus, rhinovirus, polio virus, hepatitis A virus, bovine viral diarrhea virus or Japanese encephalitis virus which comprises administering to the subject a therapeutically effective amount of the compound according to claim
 1. 4. The method of treatment according to claim 3 wherein the subject is infected with hepatitis C virus.
 5. A method of treatment in a subject in need thereof, which comprises contacting at least one hepatitis C virus infected cell with at least one compound according to claim
 1. 6. A compound represented by formula I, or a stereoisomer thereof:

wherein Z is

R¹ is hydrogen or phenyl; R² is hydrogen; R^(3a) is hydrogen; R^(3b) is CH₃; R⁴ is hydrogen, ^(i)Pr, or cyclopentyl; R⁵ is hydrogen; R⁶ is CH₃; X is F; R⁸ is OMe, —N(—CH₂CH₂CH₂—), OBn, or OH; and R⁹ is NH₂.
 7. A pharmaceutical composition comprising a pharmaceutically acceptable medium and the compound according to claim
 6. 8. A method of treatment of a subject infected with a virus selected from the group consisting of hepatitis C virus, West Nile virus, yellow fever virus, dengue virus, rhinovirus, polio virus, hepatitis A virus, bovine viral diarrhea virus or Japanese encephalitis virus which comprises administering to the subject a therapeutically effective amount of the compound according to claim
 6. 9. The method of treatment according to claim 8 wherein the subject is infected with hepatitis C virus.
 10. A method of treatment in a subject in need thereof, which comprises contacting at least one hepatitis C virus infected cell with at least one compound according to claim
 6. 11. The compound of claim 1 wherein R⁸ is OMe.
 12. The compound of claim 1 wherein R⁸ is OH.
 13. A pharmaceutical composition comprising a pharmaceutically acceptable medium and the compound according to claim
 11. 14. A pharmaceutical composition comprising a pharmaceutically acceptable medium and the compound according to claim
 12. 15. A method of treatment of a subject infected with a virus selected from the group consisting of hepatitis C virus, West Nile virus, yellow fever virus, dengue virus, rhinovirus, polio virus, hepatitis A virus, bovine viral diarrhea virus or Japanese encephalitis virus which comprises administering to the subject a therapeutically effective amount of the compound according to claim
 11. 16. The method of treatment according to claim 15 wherein the subject is infected with hepatitis C virus.
 17. A method of treatment in a subject in need thereof, which comprises contacting at least one hepatitis C virus infected cell with at least one compound according to claim
 11. 18. A method of treatment of a subject infected with a virus selected from the group consisting of hepatitis C virus, West Nile virus, yellow fever virus, dengue virus, rhinovirus, polio virus, hepatitis A virus, bovine viral diarrhea virus or Japanese encephalitis virus which comprises administering to the subject a therapeutically effective amount of the compound according to claim
 12. 19. The method of treatment according to claim 18 wherein the subject is infected with hepatitis C virus.
 20. A method of treatment in a subject in need thereof, which comprises contacting at least one hepatitis C virus infected cell with at least one compound according to claim
 12. 